Total RNA was extracted from mice tissues or primary cultural cells using the RNeasy Lipid Tissue Mini Kit (Qiagen, Germantown, MD, USA). The purity and concentration of total RNA were determined by a NanoDrop spectrophotometer (Thermo Fisher Scientific). One μg of total RNA was reverse-transcribed using a cDNA kit (AB Applied Biosystems, Waltham, MA, USA). Real-time PCR amplification was detected using SYBR Green PCR master mixture (Qiagen) on a Roche 480 Real-time PCR system (Basel, Switzerland). The primer sequences used in real-time PCR are shown in Supplementary Table S1 . Western blot analysis was performed following the procedures described previously [45 (link)].
480 real time pcr system
Manufactured by Roche
Sourced in Switzerland, Japan, United States, Germany, China, Sweden
The Roche 480 real-time PCR system is a laboratory instrument designed for the amplification and detection of nucleic acid sequences through real-time polymerase chain reaction (PCR) technology. It enables the quantification of target DNA or RNA molecules in a sample with high sensitivity and precision.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (19 mentions)
Trizol, by Thermo Fisher Scientific (8 mentions)
Sybr premix ex taq 2, by Takara Bio (8 mentions)
Primescript rt reagent kit, by Takara Bio (7 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (4 mentions)
Sybr green pcr kit, by Takara Bio (4 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Trizol agent, by Thermo Fisher Scientific (3 mentions)
Hairpin ittm mirnas qpcr quantitation assay kit, by GenePharma (3 mentions)
Sybr green 1 master mix, by Vazyme (3 mentions)
Rneasy lipid tissue mini kit, by Qiagen (2 mentions)
Reverse transcription system, by Toyobo (2 mentions)
Lightcycler 480 sybr green 1 master, by Roche (2 mentions)
Faststart essential dna green master kit, by Roche (2 mentions)
Primescript 2 first strand cdna synthesis kit, by Takara Bio (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480 sybr green 1 master mix, by Roche (2 mentions)
66 protocols using 480 real time pcr system
1
Total RNA Extraction and Quantification
2
Quantification of miR-654-5p Expression
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Total RNAs were extracted using the TRIzol agent (Ambion, ThermoFisher Scientific, Waltham, MA, USA) according to the instruction of the manufacturer. The reverse transcription of RNA and quantitative real-time PCR was performed using the Hairpin-itTM miRNAs qPCR Quantitation Assay Kit (GenePharma, Shanghai, China) according to the manufacturer’s instructions. Quantitative RT-PCR was performed using a Roche 480 real-time PCR system. The 2−ΔΔCt method was used to evaluate miR-654-5p gene expression after normalization for expression of the endogenous controls U6 (U6 non-coding small nuclear RNA). All primers for miR-654-5p and the U6 genes were designed, synthesized, and verified using GenePharma. Mature miR-654-5p sequence was as follows: 5′-UGGUGGGCCGCAGAACAUGUGC-3′. Each experiment was repeated at least three times.
3
Quantification of Verticillium dahliae Biomass
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Quantification of V. dahliae biomass was performed in accordance with previously described methods [31 (link)]. The 1-cm stem sections above the cotyledon node were ground to a powder, and an aliquot of approximately 100 mg was used for DNA isolation [32 (link)]. Quantitative real-time PCR was conducted using a Roche 480 real-time PCR system (Roche, Basle, Sweden). To measure the V. dahliae biomass, the internal transcribed spacer region of the ribosomal DNA was targeted using the fungus-specific ITS1-F primer [33 (link)] in combination with the V. dahliae-specific reverse primer ST-VE1-R [34 (link)], generating a 200-bp amplicon. The average fungal biomass was determined using at least five Vd991-inoculated plants for each line. The relative fold changes of the target genes were calculated as described [35 (link)]. The reference gene was the cotton ubiquitin 7 (UBQ7) gene. The primers used for PCR amplification are listed in Table S1 .
4
Serum miRNA RT-qPCR Protocol
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Reverse transcription with 7 μL of RNA extracted from serum sample was performed using PrimeScript RT Enzyme System, following the manufacturer's protocol (TaKaRa, Japan). cDNA obtained from the RT reaction was diluted 1:10 with RNase-free water. The PCR reaction was performed with gene-specific primers whose sequences are described in (Supplementary Material ) using SYBR Green I PCR Master Mix (TaKaRa, Japan) in the Roche 480 Real-Time PCR System (Roche, Switzerland). Each reaction was performed in a 20 μL volume containing 2 μL cDNA, 3 μL forward primer, 1.4 μL universal reverse primer and 10 μL Power SYBR Green I PCR Master Mix. The thermal cycling profile for PCR was set up as follows: pre-denaturation at 95°Cfor 5 min, followed by 40 cycles of denaturation for 15 s at 95°C, and annealing and extension for 1 min at 60 °C. A melt curve analysis was performed at the end of each PCR cycle to validate the PCR product specificity. The detection limits and dynamic range of the RT-qPCR assay were assessed with data analyzed. Meanwhile, the absolute concentration of target miRNA was calculated using a calibration curve established with the corresponding synthetic miRNA oligonucleotide (Supplementary Material ). Each reaction was carried out in triplicate.
5
Quantification of PbPIN Gene Expression
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Total RNA was extracted using the RNAprep Pure Plant kit (Tiangen, Beijing, China), following the manufacturer’s instructions. cDNAs were synthesized from moderate total RNA using a PrimeScriptTM RT reagent kit with gDNA Eraser (TaKaRa, Tokyo, Japan). qRT-PCR was performed using the Roche 480 real-time PCR system at the standard mode with the FastStart Essential DNA Green Master kit. All reactions were performed in triplicate at a volume of 20 μL, containing 1 μL of diluted cDNA, and the pear actin gene was used as an internal control. The qRT- PCR amplification was carried out as follows: 95 °C for 5 min, 45 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. PbPIN gene-specific primers for quantitative PCR are listed in the Table 4 . The relative expression levels of PbPINs were calculated using the 2−ΔΔCT method. All qPCR analyses have three biological replicates and two technical replicates.
6
RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted using a plant RNA MIDI kit (Life-Feng, Shanghai, China). First-strand complementary DNA (cDNA) was synthesized with a Reverse Transcription system (Toyobo, Osaka, Japan) and was used as the template for quantitative real-time polymerase chain reaction (RT-qPCR) analyses along with 2 × SYBR Green I master mix (Vazyme, Nanjing, China). The RT-qPCR analyses were performed on a Roche 480 real-time PCR system (Roche, Mannheim, Germany). The RNA levels were calculated as described by Livak and Schmittgen (2001) (link). The reference gene was ACTIN8 (AT1G49240).
7
Total RNA Extraction and RT-qPCR Analysis
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The total RNA was extracted and purified using an RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. RNA quality was verified by spectrophotometric measurement on a NanoDrop 2000C instrument (Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA was synthesized using a HiScript II first Strand cDNA Synthesis Kit (Vazyme, Beijing, China) according to the manufacturer’s instructions. Reverse-transcription quantitative PCR was conducted using a Roche 480 real-time PCR system (Basil, Switzerland) in standard mode with a FastStart Essential DNA Green Master kit with the following cycling parameters: 95 °C for 5 min and 45 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. All of the reactions were performed in triplicate in a 20-µL volume containing 2 µL of 10-fold diluted cDNA, with the pear Actin gene used as the internal control. All primers are listed in Table S1 .
8
RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted with a High Purity Total RNA Rapid Extraction Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized using HiScript® II Q RT SuperMix for qPCR (+ gDNA wiper) Kit (Vazyme, Nanjing, China). Real-time quantitative PCR (RT-qPCR) was performed using AceQ® qPCR SYBR Green Master Mix Kit (Vazyme, Nanjing, China) and Roche 480 Real-Time PCR System following the manufacturer’s instructions. The rice OsActin gene (LOC_Os03g50885) was used as an internal control and for the normalization in the analysis. The relative gene expression level was calculated using the 2−ΔΔCt method as previously reported (Gao et al. 2019 (link)). The data were presented as the mean ± SD of three replicates. The primers for RT-sqPCR and RT-qPCR are listed in Additional file 3 : Table S1.
9
Quantitative Analysis of Heat Shock Proteins
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Total RNA of the samples from each treatment was extracted and reverse transcribed as described above. Primers were designed based on the conserved regions of the hsps, and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was used as the control (S1 Table ). The real-time PCR reaction was performed in a 10 μL reaction volume following the manufacturer’s protocol for SYBR ® Premix Ex Taq™ (TaKaRa, Dalian, China). The expression profiles of hsps were determined on a Roche 480 Real-Time PCR System (Roche, Switzerland) under the following conditions: 95°C for 3 min, 40 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 20 s. The melting curve analysis was applied to ensure the specificity of primers at the end of the program. The relative abundance of each hsp was calculated according to the 2−ΔΔCt method [30 (link)].
10
Quantitative miR-622 Expression Analysis
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Total RNAs were extracted using the TRIzol agent (Ambion) according to the instruction of the manufacturer. Reverse transcription of RNA and quantitative real-time PCR was performed using the Hairpin-itTM miRNAs qPCR Quantitation Assay Kit (GenePharma) according to the manufacturer's instructions. Quantitative RT-PCR was performed in a Roche 480 real-time PCR system. The 2−ΔΔCt method was used to evaluate the miR-622 gene expression after normalization for expression of the endogenous controls U6 (U6 non-coding small nuclear RNA). All primers for miR-622 and the U6 genes were synthesized and approved by GenePharma. Each experiment was repeated at least three times.
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